20.109(S15): TA notes for module 1

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20.109(S15): Laboratory Fundamentals of Biological Engineering

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Contents

General notes

Scheme: Class will test 8 cloacal samples (each team prepares one sample), in order to get enough sequencing data for establishing phylogenetic trees. Last year, the two partners in each team prepared different samples, but that created some workflow issues later in the module; this year we will have them prepare duplicates of the same sample.

Key preparation:

  • Pre-run entire bird gut microbiota experiment with 1-2 cloacal samples, to become familiar with each experiment. Be sure to test bird all bird samples that have not been used in previous semesters.
  • Pre-test DNA extraction from all 8-10 samples (just through PCR and gel test). During S13 one sample consistently failed.
  • Complete the primer design project (AIV screening experiment) to be able to answer questions about that experiment.
  • If the majority of samples fail to amplify, the problem is most likely at the PCR stage. Try using all new reagents &/or adjusting [BSA].
  • While you read the wiki, test each link, and make any link descriptions bold that are not already.
  • As you read the protocols, add reagent calculations to the Dropbox Excel sheet as needed.
  • When preparing aliquots, be sure to include extra.

Towards S15 notes:

  • Cloning troubleshooting
    • The S13 cloning kit yielded many colonies with failed sequencing. In some cases just F-seq or R-seq worked, and a close look indicated that the vector was inexplicably cutting part of itself out and then religating.
    • The S14 kit yielded better sequencing runs, but just as many failed clones – this time in a different way. Many recovered inserts appeared to be primer dimers.
    • The first option to test is including gel purification, though this addition will make lab timing tight. But if it's a simple fix to close to 100% recovery…
    • The other point to keep in mind is that the initial DNA recovery is low, despite attempts to optimize time/temp/enzymes in original pilots. Often the final PCR yield is quite high, but it is also quite variable across samples.
    • Thus, it's important that the TA checks all samples through the extraction stage, and a few through the sequencing stage.
      • Looking back at S14 pilots, it turned out that fecal samples were the ones pre-tested through the sequencing stage, whereas cloacal samples were used during class.
  • S15 changes
    • PCR cleanup will be completed prior to cloning step.
    • Microsporidia screen project replaced with AIV screen (changes made to D2, D5, and D8).


Day-by-day

Day 1

Materials required:

  • Autoclave 1.7 mL and 2 mL microcentrifuge tubes.
  • 1000 uL, 200 uL, & 20 uL filter tips, 1 box per team.
  • Before aliquoting out the cloacal samples, thaw the samples for ~4h at 4°C, and then vortex each cryovial for 2 min.
  • Bird samples! At 100 μL each in a 2 mL tube
    • Use filter pipet tip to transfer and store at -80 °C -- consult instructor about which samples to prepare.
  • Aliquots of components from QiaAMP stool kit (see Excel doc)
    • Do NOT put out ethanol until after everyone is at the incubation step; we don't want them to accidentally add it too early
    • Some aliquots may be approximate (use your judgment). For example, it will be easier to pipet approximately 0.5 mL into each tube using a serological pipet than to pipet exactly 0.46 mL eight times per day, times several reagents!
    • ASL and AL must be aliquoted the day of lab (see below)
    • InhibiTEX tablets should be singly available -- pre-cut around them; otherwise it is too easy to push out two instead of one
  • Chitinase aliquots (Sigma)
    • prep at 0.02666 U/uL in 50 mM potassium phosphate

Day of Lab (R/F):

  • Thaw that day's 100 μL samples in the fridge starting just before lecture.
  • Place 4-5 ice buckets around the room (between pairs) for keeping samples chilled at first.
  • Place chitinase aliquots on ice.
  • Before lab, move samples to the ice bucket nearest the pair that will be using those particular samples (see table on M1D1 Talk page).
  • Place autoclaved tubes (1.7 mL and 2 mL), Qiagen spin columns, tablets, and column tubes at the front.
  • Place Part 1 aliquoted reagents on the front bench. Leave the Part 2 aliquots at the TA bench for now.
  • Check ASL and AL for precipitates; pre-heat at about 40-50 °C in the water bath at the TA bench if needed.--in advance
  • Place an eppendorf tube rack in the 56 °C oven to warm it up.
  • Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C. Be sure to use the insert that can accommodate 2 mL tubes.
  • Arrange the groups that take their samples out of the 56 ° at the same time to spin their samples together.
  • Either set up second heat block at 56 °C or turn on the small oven (may be easiest).
  • Qiagen flow-throughs should be disposed of in the dedicated container in the fume hood, using a conical tube as an intermediate container.

After Lab:

  • Store extraction samples at -20 °C until next time

How it went:


Day 2

Materials required:

  • None :)

Day of Lab (T/W):

  • Be familiar with design steps and able to answer student questions.

How it went:


Day 3

Materials required:

  • Their D2 DNA extractions -- thawed just before class
  • PCR components aliquoted (except polymerase)
    • Per two teams sharing, 6 rxns + 30% per component should be reasonable
  • 1000 uL, 200 uL, & 20 uL filter tips, 1 box per team.

Day of Lab (R/F):

  • Polymerase aliquots prepared last minute: 1-3 big aliquots to share, and students will come up to the front bench when they are ready
  • Place the cryo-racks for each group on their bench right before they begin the lab.
  • Place a "finished" cryo-rack beside the PCR machine for students to place their finished PCR cocktails.
    • Remind them to place their tubes at the center because the edges warm up quickly.

After Lab:

  • Remove PCR samples and store at -20 °C. Do not leave reaction in thermocycler overnight as there was a problem with evaporation during the pilot experiments.

How it went:


Day 4

Materials required:

  • Part 2, purification
    • prepare aliquots (for each student) of the Qiagen PCR cleanup kit
  • Part 3, DNA gels
    • 1.0% agarose gels
      • prep morning of or day before and cover with saran wrap
      • 1 gel per 4 groups
    • 6X loading dye aliquots at front bench
    • 1kb DNA ladder - couple shared aliquots at gel bench
  • Part 4, ligation and start transformation
    • water (>10 uL) aliquots per team (molecular biology grade, sterile)
    • vector, T4, and rxn buffer just from the kit tubes to preserve volume
    • pre-warmed LB
  • Part 5, O/N culture prep
    • tons of autoclaved glass tubes
    • LB (25 mL aliquots) and Kanamycin (chilled) up front, along with pipets and pipet-aids
  • Part 6, finish transformation
    • Add denature ethanol (has isopropanol) to the alcohol burners
    • Ensure ethanol jars are filled, relatively fresh
    • Glass spreaders and lighters available
    • All at front bench
    • 70 (at least 66) of LB-KAN plates, put out in advance so students can warm them

Day of Lab (R/F):

  • Thaw each student's 16s DNA from Day 3
  • Gel bench set up: electrophoresis equipment, DNA ladders kept cool in orange freezer boxes.
  • Fill heat block with water and set to 42 °C.
  • Place the LB aliquots at 37 °C to warm them up.
  • Place an ice bucket for each team containing the ligation reagents, DNA samples + Kanamycin.
  • Keep competent cells at -80C... when students need them, give them an aliquot.
  • Autoclaved 1.5 mL eppendorf tubes at front bench.

After Lab: Keep LB+Kan test tubes in the fridge until next day.

Next day:

  1. Check plates for colonies. Use plates from pilot experiments or plates from another student with the same bird sample if there are no/too few colonies.
  2. Inoculate 8 colonies per person. Make sure the roller is on!

How it went:


Day 5

Materials required:

  • Part 1: mini prep
    • To keep the day running on time, prep individual aliquots per team
    • Omega kit way cheaper than Qiagen kit
    • Have available sterile water taken to pH 8.5 with sterile NaOH
  • Part 2: spec DNA, materials at front bench
    • Cuvettes
    • Plenty of MBG water
  • Part 3: sequencing rxns, at front bench
    • Plates and strip caps, plus location table printed out for students to check off
    • Clearly labeled sequencing primer -- F and R -- available, along with labeled reservoirs
    • Sterile water aliquots per team
  • Part 4: AIV screen primer prep
    • Again, MBG sterile water -- can be one aliquot for Parts 3+4 together
    • Aliquots of cDNA
      • Two positive and one negative sample per group
      • Group will need enough cDNA to complete 4 reactions per sample (round to 4.5)

Day of Lab (T/W):

  • Remove O/N cultures from incubator in the morning, place at front bench
  • Place plates out for them to count their colonies

After Lab:

  • Prepare an additional 8 colonies per bird to increase odds of having enough sequencing results for phylogenetic analysis
  • Keep an eye on sequencing results as they come in. Prepare additional minipreps etc. as needed for bird samples with high number of failures.

How it went:

Day 6

Day of Lab (R/F):

  • Help prepare journal club room (16-336) in advance.
  • Feel free to participate in Q&A sessions of the talks.

Day 7

Materials required:

  • None

Day of Lab (T/W):

  • Be familiar with analysis steps and able to answer student questions.

How it went:


Day 8

Materials required:

  • None

Day of Lab (T/W):

  • Be familiar with analysis steps and able to answer student questions.

How it went:


Day 9

Day of Lab (T/W):

  • Help prepare journal club room (16-336) in advance.
  • Feel free to participate in Q&A sessions of the talks.
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